EB 病毒Rta 优势表位抗原的克隆表达及在应用ELISA 诊断鼻咽癌中的价值

目的　制备高活性EB 病毒立即早期蛋白Rta 优势表位抗原，并初步评价该抗原在鼻咽癌（nasopharyngeal carcinoma, NPC）诊断中的价值。方法　分析Rta 氨基酸序列，选取抗原优势表位Rta（301~440aa），合成目的基因， 连接pBVGST-6His 载体，构建重组pBV-Rta 表达质粒，进行诱导表达获得纯化抗原。采用间接ELISA 初步评价优势表 位抗原在鼻咽癌诊断中的价值，以纯化的pBV-Rta 优势表位抗原为包被抗原，抗人IgA，IgG 和IgM 为二抗，检测由烟 台毓璜顶医院提供的50 例经临床病理确诊的NPC 患者血清样本和90 例健康体检者血清样本。结果　获得了高效原核 表达的pBV-Rta 优势表位抗原(301~440aa)，经过小样本验证，确定pBV-Rta 具有良好的抗原活性和特异度。放大样本 进行检测，基于Rta 优势表位抗原的Rta-IgG 间接ELISA 方法检测灵敏度和特异度分别为73.08% 和95.79%。Rta-IgG 检测的AUC 值为0.860。结论　Rta 优势表位抗原pBV-Rta 是优选的可以用于Rta-IgG 检测的抗原，利用该抗原所建立 的间接ELISA 检测方法可以很好地区分鼻咽癌患者和健康对照。

Cloning and Expression of Dominant Epitope Antigen of Epstein-Barr Virus Rta Protein and Its Application in the Diagnosis of Nasopharyngeal Carcinoma by ELISA

Objective　To prepare the dominant epitope antigen of Rta of Epstein Barr virus (EBV) immediate early protein, and evaluate its value in the diagnosis of nasopharyngeal carcinoma(NPC). Methods　The amino acid sequence of Rta was analyzed, and the dominant epitope antigen of Rta (301~440aa) was selected to synthesize the target gene. An expression-construct for Rta (301~440aa) was engineered by inserting the corresponding DNA into a pBVGST-6His plasmid. The recombinant pBV-Rta expression plasmid was induced expression to obtain the purified antigen.The value of the dominant epitope antigen in the diagnosis of NPC was preliminarily evaluated by indirect ELISA. The purified pBV-Rta dominant epitope antigen was used as the encapsulated antigen, and the anti-human IgA, IgG and IgM were used as the secondary antibody. The sensitivitiy and specificitiy of pBV-Rta were detected in serum of 50 NPC patients confirmed by clinicopathology and 90 healthy patients tested by Yantai Yuhuangting Hospital. Results　The domonant epitope antigen (301~440aa) of pBV-Rta was highly prokaryotic expressedin E. coli. After small sample verification, it was determined that pBV-Rta had good antigenic activity and specificity. The sensitivitiy and specificitiyof Rta-IgG were 73.08% and 95.79%, respectively, by indirect ELISA based on Rta domonant epitope antigen,and the AUC value of Rta-IgG was 0.860. Conclusion　The Rta dominant epitope antigen pBV-Rta is the preferred antigen that can be used for Rta-IgG detection. The indirect ELISA detection methods of Rta-IgG established using Rta dominant epitope antigen can be used to distinguish NPC patients from healthy controls.